The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Influenza is an acute contagious illness often characterized by inflammation of the respiratory tract, fever, chills, muscular pain, prostration and maliase and is caused by the Orthomyxoviridae family of influenza viruses. Infection can cause mild to severe illness, and at times can lead to death.
Influenza viruses are classified into three types: Types A, B, and C. Type A influenzas have been responsible for pandemics, spreading over a wide geographic area and affecting a large proportion of the population. Type A influenza viruses are known to infect many animals including birds and mammals (e.g., humans, dogs, horses, cattle, sheep, pigs and seals). In contrast, type B influenzas tend usually to infect only humans. Type A and B influenzas are responsible for the increased flu-related illnesses, hospitalizations and deaths that occur each year. Type C influenzas tend to be the least worrisome. Infection in humans may cause mild respiratory distress or no symptoms at all.
Type A influenza viruses are further classified by strain. The strain name is determined by identifying differences between two antigenic proteins, hemagglutinin (“HA”) and neuraminidase (“NA”), both present on the viral surface. Rapid alterations in the sequence of these two proteins (termed “antigenic shift”) are mainly responsible for the yearly changes in immunogenicity and the requirement for new vaccines each year. Examples of antigenic shift and the concomitant strain change are clear: between 1918 and 1957, the H1N1 strain was dominant; between 1957 and 1968, the H2N2 strain was dominant; then, since 1968, the H3N2 virus dominated. Most recently, in 1997, the H5N1 strain of avian influenza was shown to infect humans. In addition to antigenic shift, the antigenic properties of influenza viruses can also change more slowly via “antigenic drift,” the slow, gradual process of viral evolution. Antigenic variation (drift) of HA sequences is noted, for example, in human trials with the escape of viral strains from vaccine induced immunity.
One cause of the rapid sequence changes associated with “antigenic shift” is the formation of reassortant viral strains. The pig can be infected by both human and avian influenza strains, and an exchange of RNA stands between human and avian strains can occur within the pig. These “reassortant” viruses may infect the human species causing a yearly epidemic of influenza. Thus, the rapid changes in the HA and NA proteins resulting from antigenic shift render old influenza viral vaccines ineffective.
Hemagglutinin (“HA”), one of the proteins affected by antigenic shift, is an antigenic glycoprotein found on the surface of influenza viruses. HA functions to secure the virus to the target cells by binding to N-acetyl neuraminic acid or sialic acid on host cell receptors. HA is composed of two subunits, HA1 and HA2. HA2 is the viral membrane anchoring domain, while HA1 is responsible for binding host cell receptors. As noted above, HA is highly mutable and variations, mainly in HA1, are a key source of viral antigen variability conferring the ability to evade the immune system. There are currently between 16 and 20 different HA varieties known. H1, H2 and H3 have been the dominant human influenza subtypes, while the H5 and H7 subtypes have been prevalent for avian species.
Neuraminidase (“NA”) is also presented on the viral surface and functions to catalyze the removal of terminal sialic acid residues of glycosyl groups, thus destroying potential receptors for hemagglutinin. It is probable that neuraminidase is required to prevent viral aggregation and to promote more efficient spreading of the virus from cell to cell. The neuraminidase protein sequence is also highly variable; there are currently nine different neuraminidase varieties known. Accordingly, changes in this protein sequences also play a role in antigenic shift.
Another viral protein present on the viral influenza surface is the Matrix protein 2 (“M2”), an ion channel protein that selectively allows protons to enter the virus. After the virus enters a cell, an influx of protons is key in the removal of the viral protein coat. The M2 protein is homotetramer comprised of three domains: a 23 amino-acid region present on the outside of the virus (extra cellular domain), a 54 amino acid region that is inside the virus (cytoplasmic domain) and a 19 amino-acid transmembrane domain. M2 is expressed at low levels on the viral surface but is present at high levels on influenza infected cells. The M2 protein sequence is stable compared to hemagglutinin or neuraminidase. In fact, the 23-amino acid extra cellular domain of M2 is well conserved in many known influenza strains (some exceptions include A/PR/8/34, A/Brevig and Mission/1/8). Although this protein is not immunogenic normally, it has been shown that chimeric molecules made from the extra cellular domain of the M2 and “adjuvant proteins” such as the hepatitis B core protein induce a potent immune response (Virology 2005 337:149-161; Infection and Immunity 2002 70:6860-70).
The intranasal administration of the M2HB core particle, along with adjuvants (such as a detoxified enterotoxin adjuvant), protected 2-4 month old BalbC mice from challenge with human influenza virus (Virology 2005 337:149-161). M2, which is important in determining host range (J. Virology 1999 73:3366-3374), is present at such low levels in the virus that antibodies are not generated. HA has been the target for vaccines since the antibodies to HA have been shown to prevent influenza viral infection.
A commercial human vaccine against the H5N1 strain of influenza has not yet been developed, although the H5 strain of avian influenza virus was seen to infect human directly in 1997; 6/18 infected people died. Since that time, outbreaks have occurred in 2003 in Hong Kong (2 deaths in 3 cases), and in Vietnam/Thailand in 2004 (28 deaths in 39 infected cases (Kash J C et al., Journal of Virology 78: 9499-9511 (2004); Apisarnthanarak A, et al., Emerg. Infect. Dis. 10 (2004)). In 2005, 79/150 H5N1 infected human beings died. Over 99% of the sequences of the infecting H5N1 viruses are avian, suggesting direct transmission from the poultry to human beings (Science 2001 293:1840-1842; J. Virology 2000 74:1443-1450). When the avian influenza viruses acquire the capability of directly jumping from birds to humans, there is a potential for a pandemic. For this to occur, the virus must be able to be transmitted in aerosols from person to person. A transition from avian:human to human:human transmission resulting in a pandemic was documented for the first time in 1918 in an H1N1 strain. The result was more than 600,000 deaths in the USA and 40 million worldwide (J. Virology 2004 78: 9499-9511). Statistics show that most of the deaths were restricted to younger individuals living in crowded conditions (soldiers involved in World War I).
The H5N1 strain has been reported to infect humans (J. Virology 2000 74:1443-1450). It is estimated that if this virus acquires the capability of spreading from human to human, there impact in the USA will be over 200,000 deaths, over 700,000 hospitalizations, over 40 million outpatient visits, and an economic impact of over 100 billion dollars (Infect. Dis. 1999 5:659-671). The recent apparent trend for increased reporting deaths of individuals believed infected with the avian flu virus has created concern among governments around the world (J. Virology 2004 78:9499-9511; J. Emerg. Infect. Dis. 2004 10; Virology 2003 208:270-278; Eur. J. Biochem. 1999 260:166-175).
The testing of vaccine efficacy for the H5N1 avian flu has been carried out in mice and ferrets, but the virulence of the various human strains in ferrets is closer to that seen in humans (J. Virology 2005 79:2191-2198). The pathogenicity of the various strains has been correlated with the HA protein structure (Id). After the 1997 cases in Hong Kong, two strategies for vaccines against the H5N1 strains were tested. First, it was discovered that a subunit H5 vaccine did not appear to be immunogenic in humans (J. Infect. Dis. 2005 191:1213-1215; Vaccine 2003 21:1687-1693). However, the addition of MF59 adjuvant increased the antibody response (PNAS USA 2005 102:12915-12920). In a second approach, a multivalent vaccine (H3N1 and H5N1) was used but this proved equally ineffective (Virology 1999 73:2094-2098).
Recently, an HA DNA vaccine was shown to protect mice from the H5N1 strain (clintrials.gov/ct/gui/sho/NCT00110279;jsessionid=743259FCC0A680603EA), and there is currently a clinical trial to evaluate the immune response to H9N2 avian flu in humans (Vaccine 2002 20:1099-1105). The H9N2 study involves a cold-adapted resorted attenuated viral vaccine which is administered by intramuscular injection. It is hoped that this study will provide insight into an H5N1 immune response. Additionally, an intramuscularly administered vaccine from baculovirus expressing H5 HA was tested in 147 adults. A 23% antibody response was observed after a single injection and 52% response after two injections (Lancet 2004 357:1937-1943).
The elderly are especially at risk with respect to influenza infection, and vaccination against influenza is recommended for older individuals to prevent the potentially deadly complications of infection such as pneumonia or bronchitis. One cause of increased risk in the elderly is the decrease in function of the immune system with age. For example, there is a decrease in the number of naïve, antigen unexposed CD4 and CD8 T cells. Additionally, the ratio of the naïve to memory CD8/CD4 cells decreases as the chronological age increases. Further, CD4 cells become impaired, acquiring both quantitative and functional defects, such as diminished levels of the CD40 ligand (CD40L) on the surface of CD4 cells as well as a temporal retardation of the rate at which CD40 ligand (CD40L) is expressed on the surface of the CD4 cells following activation. Accordingly, the amount of antibody that an elderly system is able to generate will be lower following infection or conventional vaccination.
Testing has shown that current methods of vaccination are, at best, only moderately effective. Usually, three strains of the human influenza virus are grown up in eggs, purified and then chemically inactivated. Using the induction of neutralizing antibodies in the vaccinated individuals as an endpoint for response, the response to the vaccine is in the 65-70% range (Lancet 2004 357:1937-1943). The response is 4-fold less in individuals vaccinated after age 55.
Vaccines have been described that include an expression vector encoding a fusion protein that includes an antigen fused to CD40 ligand. See, e.g., U.S. Patent Application Publication US 2005-0226888 (application Ser. No. 11/009,533) titled “Methods for Generating Immunity to Antigen,” filed Dec. 10, 2004.